RESUMO
Calcium transfer into the mitochondrial matrix during sarcoplasmic reticulum (SR) Ca2+ release is essential to boost energy production in ventricular cardiomyocytes (VCMs) and match increased metabolic demand. Mitochondria from female hearts exhibit lower mito-[Ca2+] and produce less reactive oxygen species (ROS) compared to males, without change in respiration capacity. We hypothesized that in female VCMs, more efficient electron transport chain (ETC) organization into supercomplexes offsets the deficit in mito-Ca2+ accumulation, thereby reducing ROS production and stress-induced intracellular Ca2+ mishandling. Experiments using mitochondria-targeted biosensors confirmed lower mito-ROS and mito-[Ca2+] in female rat VCMs challenged with ß-adrenergic agonist isoproterenol compared to males. Biochemical studies revealed decreased mitochondria Ca2+ uniporter expression and increased supercomplex assembly in rat and human female ventricular tissues vs male. Importantly, western blot analysis showed higher expression levels of COX7RP, an estrogen-dependent supercomplex assembly factor in female heart tissues vs males. Furthermore, COX7RP was decreased in hearts from aged and ovariectomized female rats. COX7RP overexpression in male VCMs increased mitochondrial supercomplexes, reduced mito-ROS and spontaneous SR Ca2+ release in response to ISO. Conversely, shRNA-mediated knockdown of COX7RP in female VCMs reduced supercomplexes and increased mito-ROS, promoting intracellular Ca2+ mishandling. Compared to males, mitochondria in female VCMs exhibit higher ETC subunit incorporation into supercomplexes, supporting more efficient electron transport. Such organization coupled to lower levels of mito-[Ca2+] limits mito-ROS under stress conditions and lowers propensity to pro-arrhythmic spontaneous SR Ca2+ release. We conclude that sexual dimorphism in mito-Ca2+ handling and ETC organization may contribute to cardioprotection in healthy premenopausal females.
Assuntos
Miócitos Cardíacos , Retículo Sarcoplasmático , Ratos , Masculino , Feminino , Animais , Humanos , Idoso , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Caracteres Sexuais , Mitocôndrias/metabolismo , Sinalização do Cálcio , Cálcio/metabolismoRESUMO
PURPOSE OF REVIEW: Many mechanical load-bearing joints of the body are prone to posttraumatic osteoarthritis (PTOA), including the knee joint and temporomandibular joint (TMJ). Early detection of PTOA can be beneficial in prevention or alleviating further progression of the disease. RECENT FINDINGS: Various mouse models, similar to those used in development of novel diagnosis strategies for early stages of OA, have been proposed to study early PTOA. While many studies have focused on OA and PTOA in the knee joint, early diagnostic methods for OA and PTOA of the TMJ are still not well established. Previously, we showed that fluorescent near-infrared imaging can diagnose inflammation and cartilage damage in mouse models of knee PTOA. Here we propose that the same approach can be used for early diagnosis of TMJ-PTOA. In this review, we present a brief overview of PTOA, application of relevant mouse models, current imaging methods available to examine TMJ-PTOA, and the prospects of near-infrared optical imaging to diagnose early-stage TMJ-OA.
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Osteoartrite/diagnóstico , Animais , Diagnóstico por Imagem , Modelos Animais de Doenças , Progressão da Doença , Diagnóstico Precoce , Humanos , Camundongos , Osteoartrite/patologia , Osteoartrite do Joelho/diagnóstico , Osteoartrite do Joelho/patologia , Transtornos da Articulação Temporomandibular/diagnóstico , Transtornos da Articulação Temporomandibular/patologiaRESUMO
BACKGROUND: Fanconi anaemia (FA) is an inherited disease with bone marrow failure, variable congenital and developmental abnormalities, and cancer predisposition. With improved survival, non-haematological manifestations of FA become increasingly important for long-term management. While renal abnormalities are recognized, detailed data on patterns and frequency and implications for long-term management are sparse. METHODS: We reviewed clinical course and imaging findings of FA patients with respect to renal complications in our centre over a 25-year period to formulate some practical suggestions for guidelines for management of renal problems associated with FA. RESULTS: Thirty patients including four sibling sets were reviewed. On imaging, 14 had evidence of anatomical abnormalities of the kidneys. Two cases with severe phenotype, including renal abnormalities, had chronic kidney disease (CKD) at diagnosis. Haematopoietic stem cell transplantation was complicated by significant acute kidney injury (AKI) in three cases. In three patients, there was CKD at long-term follow-up. All patients had normal blood pressure. CONCLUSIONS: Evaluation of renal anatomy with ultrasound imaging is important at diagnostic workup of FA. While CKD is uncommon at diagnosis, our data suggests that the incidence of CKD increases with age, in particular after haematopoietic stem cell transplantation. Monitoring of renal function is essential for management of FA. Based on these long-term clinical observations, we formulate some practical guidelines for assessment and management of renal abnormalities in FA.
Assuntos
Injúria Renal Aguda/terapia , Anemia de Fanconi/terapia , Rim/anormalidades , Assistência de Longa Duração/normas , Insuficiência Renal Crônica/terapia , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/epidemiologia , Injúria Renal Aguda/etiologia , Criança , Pré-Escolar , Progressão da Doença , Anemia de Fanconi/complicações , Anemia de Fanconi/diagnóstico , Feminino , Seguimentos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Incidência , Lactente , Rim/diagnóstico por imagem , Assistência de Longa Duração/métodos , Masculino , Guias de Prática Clínica como Assunto , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/epidemiologia , Insuficiência Renal Crônica/etiologia , Estudos Retrospectivos , UltrassonografiaRESUMO
The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.
Assuntos
Lisossomos/metabolismo , Fagossomos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Receptores de Superfície Celular/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Escherichia coli/metabolismo , Feminino , Fibroblastos/metabolismo , Concentração de Íons de Hidrogênio , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Fusão de Membrana , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência , Frações Subcelulares/metabolismoRESUMO
Memory encoding sometimes must occur during a period of sleep deprivation. The question was whether one night of sleep deprivation inhibits encoding on a perceptual learning task (the texture discrimination task). The sample was 18 human participants (M age=22.1 yr., SEM=0.5; 8 men). The participants were randomized to a sleep deprivation or sleep control condition and, after the manipulation, were given two administrations of the texture discrimination task. All participants were given an opportunity for a 90 min. nap between the two administrations. Performance was measured by the interpolated stimulus-to-mask-onset asynchrony (i.e., the inter-stimulus interval), at which the percentage of correct responses for the stimuli in the participant's peripheral vision fell below 80%. Offline consolidation was defined as a decrease in this index between the two administrations. Participants who were sleep deprived prior to encoding exhibited similar offline consolidation (M=-5.3 msec., SEM=2.3) compared to participants who were not sleep deprived prior to encoding (M=-6.2 msec., SEM=3.9); the two-way interaction between time and condition was not significant. In light of reports in the literature, these results indicate encoding following sleep deprivation may be influenced by both the type of task encoded and the brain regions involved in memory processing.
Assuntos
Aprendizagem/fisiologia , Reconhecimento Visual de Modelos/fisiologia , Privação do Sono/fisiopatologia , Adulto , Feminino , Humanos , Masculino , Consolidação da Memória/fisiologia , Projetos Piloto , Adulto JovemRESUMO
BACKGROUND/AIMS: HuR is an RNA-binding protein that regulates the post-transcriptional life of thousands of cellular mRNAs and promotes cell survival. HuR is expressed as two mRNA transcripts that are differentially regulated by cell stress. The goal of this study is to define factors that promote transcription of the longer alternate form. METHODS: Effects of transcription factors on HuR expression were determined by inhibition or overexpression of these factors followed by competitive RT-PCR, gel mobility shift, and chromatin immunoprecipitation. Transcription factor expression patterns were identified through competitive RT-PCR and Western analysis. Stress responses were assayed in thapsigargin-treated proximal tubule cells and in ischemic rat kidney. RESULTS: A previously described NF-κB site and a newly identified Sp/KLF factor binding site were shown to be important for transcription of the long HuR mRNA. KLF8, but not Sp1, was shown to bind this site and increase HuR mRNA levels. Cellular stress in cultured or native proximal tubule cells resulted in a rapid decrease of KLF8 levels that paralleled those of the long HuR mRNA variant. CONCLUSIONS: These results demonstrate that KLF8 can participate in regulating expression of alternate forms of HuR mRNA along with NF-κB and other factors, depending on cellular contexts.
Assuntos
Proteínas ELAV/fisiologia , Proteínas Repressoras/fisiologia , Animais , Sequência de Bases , Western Blotting , Imunoprecipitação da Cromatina , Primers do DNA , Proteínas ELAV/genética , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/fisiologia , Células LLC-PK1 , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Ratos , SuínosRESUMO
Vacuolar ATPases (V-ATPases) are highly conserved proton pumps that regulate organelle pH. Epithelial luminal pH is also regulated by cAMP-dependent traffic of specific subunits of the V-ATPase complex from endosomes into the apical membrane. In the intestine, cAMP-dependent traffic of cystic fibrosis transmembrane conductance regulator (CFTR) channels and the sodium hydrogen exchanger (NHE3) in the brush border regulate luminal pH. V-ATPase was found to colocalize with CFTR in intestinal CFTR high expresser (CHE) cells recently. Moreover, apical traffic of V-ATPase and CFTR in rat Brunner's glands was shown to be dependent on cAMP/PKA. These observations support a functional relationship between V-ATPase and CFTR in the intestine. The current study examined V-ATPase and CFTR distribution in intestines from wild-type, CFTR(-/-) mice and polarized intestinal CaCo-2BBe cells following cAMP stimulation and inhibition of CFTR/V-ATPase function. Coimmunoprecipitation studies examined V-ATPase interaction with CFTR. The pH-sensitive dye BCECF determined proton efflux and its dependence on V-ATPase/CFTR in intestinal cells. cAMP increased V-ATPase/CFTR colocalization in the apical domain of intestinal cells and redistributed the V-ATPase Voa1 and Voa2 trafficking subunits from the basolateral membrane to the brush border membrane. Voa1 and Voa2 subunits were localized to endosomes beneath the terminal web in untreated CFTR(-/-) intestine but redistributed to the subapical cytoplasm following cAMP treatment. Inhibition of CFTR or V-ATPase significantly decreased pHi in cells, confirming their functional interdependence. These data establish that V-ATPase traffics into the brush border membrane to regulate proton efflux and this activity is dependent on CFTR in the intestine.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Enterócitos/metabolismo , ATPases Vacuolares Próton-Translocadoras/fisiologia , Animais , Células CACO-2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvilosidades/metabolismo , Transporte Proteico/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Human antigen R (HuR) is a post-transcriptional regulator of gene expression that plays a key role in stabilizing mRNAs during cellular stress, leading to enhanced survival. HuR expression is tightly regulated through multiple transcription and post-transcriptional controls. Although HuR is known to stabilize a subset of mRNAs involved in cell survival, its role in the survival pathway of PI3-kinase/Akt signaling is unclear. Here, we show that in renal proximal tubule cells, HuR performs a central role in cell survival by amplifying Akt signaling in a positive feedback loop. Key to this feedback loop is HuR-mediated stabilization of mRNA encoding Grb10, an adaptor protein whose expression is critical for Akt activation. Stimulation of Akt by interaction with Grb10 then activates NF-κB, which further enhances HuR mRNA and protein expression. This feedback loop is active in unstressed cells, but its effects are increased during stress. Therefore, this study demonstrates a central role for HuR in Akt signaling and reveals a mechanism by which modest changes in HuR levels below or above normal may be amplified, potentially resulting in cell death or cellular transformation.
Assuntos
Apoptose/fisiologia , Proteínas ELAV/metabolismo , Retroalimentação Fisiológica/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Proteínas ELAV/genética , Proteína Adaptadora GRB10/genética , Proteína Adaptadora GRB10/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , NF-kappa B , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Reação em Cadeia da Polimerase , Análise Serial de Proteínas , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SuínosRESUMO
Introduction: Myosin IXB (MYO9B) is an unconventional myosin with RhoGAP activity and thus is a regulator of actin cytoskeletal organization. MYO9B was previously shown to be necessary for skeletal growth and health and to play a role in actin-based functions of both osteoblasts and osteoclasts. However, its role in responses to mechanical stimulation of bone cells has not yet been described. Therefore, experiments were undertaken to determine the role of MYO9B in bone cell responses to mechanical stress both in vitro and in vivo. Methods: MYO9B expression was knocked down in osteoblast and osteocyte cell lines using RNA interference and the resulting cells were subjected to mechanical stresses including cyclic tensile strain, fluid shear stress, and plating on different substrates (no substrate vs. monomeric or polymerized collagen type I). Osteocytic cells were also subjected to MYO9B regulation through Slit-Robo signaling. Further, wild-type or Myo9b -/- mice were subjected to a regimen of whole-body vibration (WBV) and changes in bone quality were assessed by micro-CT. Results: Unlike control cells, MYO9B-deficient osteoblastic cells subjected to uniaxial cyclic tensile strain were unable to orient their actin stress fibers perpendicular to the strain. Osteocytic cells in which MYO9B was knocked down exhibited elongated dendrites but were unable to respond normally to treatments that increase dendrite length such as fluid shear stress and Slit-Robo signaling. Osteocytic responses to mechanical stimuli were also found to be dependent on the polymerization state of collagen type I substrates. Wild-type mice responded to WBV with increased bone tissue mineral density values while Myo9b -/- mice responded with bone loss. Discussion: These results demonstrate that MYO9B plays a key role in mechanical stress-induced responses of bone cells in vitro and in vivo.
RESUMO
One-third of postmenopausal women experience at least one osteoporotic bone fracture in their lifetime that occurs spontaneously or from low-impact events. However, osteoporosis-associated jaw bone fractures are extremely rare. It was also observed that jaw bone marrow stem cells (BMSCs) have a higher capacity to form mineralized tissues than limb BMSCs. At present, the underlying causes and mechanisms of variations between jaw bone and limb bone during postmenopause are largely unknown. Thus, the objective of the current study was to examine the site-specific effects of estrogen deficiency using comprehensive analysis of bone quantity and quality, and its association with characterization of cellular components of bone. Nine rats (female, 6 months old) for each bilateral sham and ovariectomy (OVX) surgery were obtained and maintained for 2 months after surgery. A hemi-mandible and a femur from each rat were characterized for parameters of volume, mineral density, cortical and trabecular morphology, and static and dynamic mechanical analysis. Another set of 5 rats (female, 9 months old) was obtained for assays of BMSCs. Following cytometry to identify BMSCs, bioassays for proliferation, and osteogenic, adipogenic, chondrogenic differentiation, and cell mitochondrial stress tests were performed. In addition, mRNA expression of BMSCs was analyzed. OVX decreased bone quantity and quality (mineral content, morphology, and energy dissipation) of femur while those of mandible were not influenced. Cellular assays demonstrated that mandible BMSCs showed greater differentiation than femur BMSCs. Gene ontology pathway analysis indicated that the mandibular BMSCs showed most significant differential expression of genes in the regulatory pathways of osteoblast differentiation, SMAD signaling, cartilage development, and glucose transmembrane transporter activity. These findings suggested that active mandibular BMSCs maintain bone formation and mineralization by balancing the rapid bone resorption caused by estrogen deficiency. These characteristics likely help reduce the risk of osteoporotic fracture in postmenopausal jawbone.
Assuntos
Células-Tronco Mesenquimais , Animais , Densidade Óssea , Osso e Ossos , Diferenciação Celular , Estrogênios , Feminino , Humanos , Osteogênese , Ovariectomia , Ratos , Células-TroncoRESUMO
AIMS: Evidence suggests alterations of thyroid hormone levels can disrupt normal bone development. Most data suggest the major targets of thyroid hormones to be the Htra1/Igf1 pathway. Recent discovery by our group suggests involvement of targets WNT pathway, specifically overexpression of antagonist Sfrp4 in the presence of exogenous thyroid hormone. MAIN METHODS: Here we aimed to model these interactions in vitro using primary and isotype cell lines to determine if thyroid hormone drives increased Sfrp4 expression in cells relevant to craniofacial development. Transcriptional profiling, bioinformatics interrogation, protein and function analyses were used. KEY FINDINGS: Affymetrix transcriptional profiling found Sfrp4 overexpression in primary cranial suture derived cells stimulated with thyroxine in vitro. Interrogation of the SFRP4 promoter identified multiple putative binding sites for thyroid hormone receptors. Experimentation with several cell lines demonstrated that thyroxine treatment induced Sfrp4 expression, demonstrating that Sfrp4 mRNA and protein levels are not tightly coupled. Transcriptional and protein analyses demonstrate thyroid hormone receptor binding to the proximal promoter of the target gene Sfrp4 in murine calvarial pre-osteoblasts. Functional analysis after thyroxine hormone stimulation for alkaline phosphatase activity shows that pre-osteoblasts increase alkaline phosphatase activity compared to other cell types, suggesting cell type susceptibility. Finally, we added recombinant SFRP4 to pre-osteoblasts in combination with thyroxine treatment and observed a significant decrease in alkaline phosphatase positivity. SIGNIFICANCE: Taken together, these results suggest SFRP4 may be a key regulatory molecule that prevents thyroxine driven osteogenesis. These data corroborate clinical findings indicating a potential for SFRP4 as a diagnostic or therapeutic target for hyperostotic craniofacial disorders.
Assuntos
Fosfatase Alcalina , Tiroxina , Camundongos , Animais , Tiroxina/metabolismo , Fosfatase Alcalina/metabolismo , Osteoblastos/metabolismo , Via de Sinalização Wnt/genética , Osteogênese/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
Osteoclasts resorb bone through the formation of a unique attachment structure called the sealing zone. In this study, a role for thyroid hormone receptor-interacting protein 6 (TRIP6) in sealing zone formation and osteoclast activity was examined. TRIP6 was shown to reside in the sealing zone through its association with tropomyosin 4, an actin-binding protein that regulates sealing dimensions and bone resorptive capacity. Suppression of TRIP6 in mature osteoclasts by RNA interference altered sealing zone dimensions and inhibited bone resorption, whereas overexpression of TRIP6 increased the sealing zone perimeter and enhanced bone resorption. Treatment of osteoclasts with lysophosphatidic acid (LPA), which phosphorylates TRIP6 at tyrosine 55 through a c-Src-dependent mechanism, caused increased association of TRIP6 with the sealing zone, as did overexpression of a TRIP6 cDNA bearing a phosphomimetic mutation at tyrosine 55. Further, LPA treatment caused increases in osteoclast fusion, sealing zone perimeter, and bone resorptive capacity. In contrast, overexpression of TRIP6 containing a nonphosphorylatable amino acid residue at position 55 severely diminished sealing zone formation and bone resorption and suppressed the effects of LPA on the cytoskeleton. LPA effects were mediated through its receptor isoform LPA(2), as indicated by treatments with receptor-specific agonists and antagonists. Thus, these studies suggest that TRIP6 is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Reabsorção Óssea/metabolismo , Osteoclastos/fisiologia , Proteínas Tirosina Quinases/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Proteína Tirosina Quinase CSK , Fusão Celular , Proteínas com Domínio LIM , Lisofosfolipídeos/farmacologia , Camundongos , Fosforilação , Complexo de Endopeptidases do Proteassoma , Quinases da Família srcRESUMO
Osteoclasts use actin-rich attachment structures in place of focal adhesions for adherence to bone and non-bone substrates. On glass, osteoclasts generate podosomes, foot-like processes containing a core of F-actin and regulatory proteins that undergo high turnover. To facilitate bone resorption, osteoclasts generate an actin-rich sealing zone composed of densely packed podosome-like units. Patterning of both podosomes and sealing zones is dependent upon an intact microtubule system. A role for unconventional myosin X (Myo10), which can bind actin, microtubules, and integrins, was examined in osteoclasts. Immunolocalization showed Myo10 to be associated with the outer edges of immature podosome rings and sealing zones, suggesting a possible role in podosome and sealing zone positioning. Further, complexes containing both Myo10 and beta-tubulin were readily precipitated from osteoclasts lysates. RNAi-mediated suppression of Myo10 led to decreased cell and sealing zone perimeter, along with decreased motility and resorptive capacity. Further, siRNA-treated cells could not properly position podosomes following microtubule disruption. Osteoclasts overexpressing dominant negative Myo10 microtubule binding domains (MyTH4) showed a similar phenotype. Conversely, overexpression of full-length Myo10 led to increased formation of podosome belts along with larger sealing zones and enhanced bone resorptive capacity. These studies suggest that Myo10 plays a role in osteoclast attachment and podosome positioning by direct linkage of actin to the microtubule network.
Assuntos
Microtúbulos/metabolismo , Miosinas/fisiologia , Osteoclastos/metabolismo , Actinas/química , Animais , Reabsorção Óssea , Linhagem Celular , Citoesqueleto/metabolismo , Genes Dominantes , Camundongos , Miosinas/química , Fenótipo , Estrutura Terciária de Proteína , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Human antigen R (HuR) is an RNA-binding protein with protective activities against cellular stress. This study considers the mechanisms by which HuR transcriptional regulation occurs in renal proximal tubule cells. Under basal conditions, HuR mRNA is expressed in two forms: one that contains a approximately 20-base 5'-untranslated region (UTR) sequence and one that contains a approximately 150-base, G+C-rich 5'-UTR that is inhibitory to translation. Recovery from cellular stresses such as thapsigargin and ATP depletion induced increased expression of the shorter, more translatable transcript and decreased expression of the longer form. Analysis of HuR upstream regions revealed sequences necessary for regulation of the shorter mRNA. Within the long, G+C-rich 5'-UTR exist multiple copies of the alternate Smad 1/5/8-binding motif GCCGnCGC. Recovery from ATP depletion induced increases in Smad 1/5/8 levels; further, gel shift and chromatin immunoprecipitation analyses demonstrated the ability of these Smads to bind to the relevant motif in the HuR 5'-UTR. Transfection of exogenous Smad 1 increased HuR mRNA expression. Finally, HuR mRNA expression driven by the Smad-binding sites was responsive to BMP-7, a protein with known protective effects against ischemic injury in kidney. These data suggest that transcriptional induction of a readily translatable HuR mRNA may be driven by a mechanism known to protect the kidney from injury and provides a novel pathway through which administration of BMP-7 may attenuate renal damage.
Assuntos
Antígenos de Superfície/genética , Proteína Morfogenética Óssea 7/farmacologia , Proteínas de Ligação a RNA/genética , Transcrição Gênica/efeitos dos fármacos , Regiões 5' não Traduzidas/genética , Trifosfato de Adenosina/metabolismo , Animais , Composição de Bases/genética , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Proteína Smad8/genética , Proteína Smad8/metabolismo , Suínos , Transcrição Gênica/genéticaRESUMO
Estrogen deficiency activates bone resorbing cells (osteoclasts) and to a lesser extent bone forming cells (osteoblasts), resulting in a gap between resorption and formation that leads to a net loss of bone. These cell activities alter bone architecture and tissue composition. Thus, the objective of this study is to examine whether multiscale (10-2 to 10-7 m) characterization can provide more integrated information to understand the effects of estrogen deficiency on the fracture risk of bone. This is the first study to examine the effects of estrogen deficiency on multiscale characteristics of the same bone specimen. Sprague-Dawley female rats (6 months old) were obtained for a bilateral ovariectomy (OVX) or a sham operation (sham). Micro-computed tomography of rat femurs provided bone volumetric, mineral density, and morphological parameters. Dynamic mechanical analysis, static elastic and fracture mechanical testing, and nanoindentation were also performed using the same femur. As expected, the current findings indicate that OVX reduces bone quantity (mass and bone mineral density) and quality (morphology, and fracture displacement). Additionally, they demonstrated reductions in amount and heterogeneity of tissue mineral density (TMD) and viscoelastic properties. The current results validate that multiscale characterization for the same bone specimen can provide more comprehensive insights to understand how the bone components contributed to mechanical behavior at different scales.
Assuntos
Densidade Óssea , Fêmur , Animais , Feminino , Fêmur/diagnóstico por imagem , Humanos , Ovariectomia , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
Postmenopausal osteoporosis causes severe loss of bone quantity and quality in limb bone but has a lesser effect on jaw bone. Thus, the objective of this study was to examine whether ovariectomy (OVX) and mastication alter the regional variation of jaw bone characteristics. Sprague-Dawley female rats (6 months) were given a bilateral OVX or a sham operation (SHAM) (n = 10 for each group). After 2 months post-OVX, the hemi-mandible from each rat was dissected. A micro-computed tomography based mean, standard deviation (SD), the lower and upper 5th percentile (Low5 and High5) values of tissue mineral density (TMD) histograms were assessed for whole bone (WB), alveolar bone (AB), cortical bone (CB), and trabecular bone (TB) regions. Morphology of TB and periodontal ligament (PDL) was also obtained. Layers of AB were segmented up to 400 µm from the PDL. Mechanical properties at the tissue level were measured by nanoindentation at the same site by a single loading-unloading cycle of indentation in hydration. The AB and TB regions had significantly lower TMD Mean, Low5, and High5 but higher SD than the CB region for both sham and OVX groups (p < 0.01). TMD parameters of the OVX group rapidly increased up to 60 µm away from the PDL and were significantly higher than those of the sham group starting at 280 µm and farther in the CB region (p < 0.05). All values of morphological and nanoindentation parameters were not significantly different between sham and OVX groups (p > 0.06). Estrogen deficiency induced by OVX did not deteriorate bone characteristics including mineral density, morphology, and nanoindentation parameters in rat mandibles. Masticatory loading had an effect on the TMD parameters at the limited region of AB. These results provide insight into why osteoporosis-associated jaw bone fractures are extremely rare.
Assuntos
Densidade Óssea , Mandíbula , Animais , Feminino , Humanos , Mandíbula/diagnóstico por imagem , Ovariectomia , Ratos , Ratos Sprague-Dawley , Microtomografia por Raio-XRESUMO
OBJECTIVE: Despite the clinical importance of chronic and severe irritability, there is a paucity of controlled trials for its pharmacological treatment. Here, we examine the effects of adding citalopram (CTP) to methylphenidate (MPH) in the treatment of chronic severe irritability in youth using a double-blind randomized placebo-controlled design. METHOD: After a lead-in phase of open treatment with stimulant, 53 youth meeting criteria for severe mood dysregulation (SMD) were randomly assigned to receive CTP or placebo (PBO) for 8 weeks. A total of 49 participants, 48 of them (98%) meeting disruptive mood dysregulation disorder (DMDD) criteria, were included in the intent-to-treat analysis. The primary outcome measure was the proportion of response based on improvements of irritability at the week 8 of the trial. RESULTS: At the end of the trial, a significantly higher proportion of response was seen in those participants randomly assigned to CTP+MPH compared to PBO+MPH (35% CTP+MPH versus 6% PBO+MPH; odds ratio = 11.70, 95% CI = 2.00-68.16, p = 0.006). However, there were no differences in functional impairment between groups at the end of the trial. No differences were found in any adverse effect between treatment groups, and no trial participant exhibited hypomanic or manic symptoms. CONCLUSION: Adjunctive CTP might be efficacious in the treatment of chronic severe irritability in youth resistant to stimulant treatment alone. CLINICAL TRIAL REGISTRATION INFORMATION: A Controlled Trial of Serotonin Reuptake Inhibitors Added to Stimulant Medication in Youth With Severe Mood Dysregulation; https://clinicaltrials.gov; NCT00794040.
Assuntos
Estimulantes do Sistema Nervoso Central , Metilfenidato , Adolescente , Transtornos de Deficit da Atenção e do Comportamento Disruptivo/tratamento farmacológico , Estimulantes do Sistema Nervoso Central/efeitos adversos , Citalopram/efeitos adversos , Método Duplo-Cego , Humanos , Humor Irritável , Metilfenidato/efeitos adversos , Resultado do TratamentoRESUMO
The RNA-binding protein human antigen R (HuR) participates in the posttranscriptional regulation of mRNAs bearing 3' AU-rich and U-rich elements, which HuR can stabilize under conditions of cellular stress. Using the LLC-PK(1) proximal tubule cell line model, we recently suggested a role for HuR in protecting kidney epithelia from injury during ischemic stress (Jeyaraj S, Dakhlallah D, Hill SR, Lee BS. J Biol Chem 280: 37957-37964, 2005; Jeyaraj SC, Dakhlallah D, Hill SR, Lee BS. Am J Physiol Renal Physiol 291: F1255-F1263, 2006). Here, we have extended this work to show that small interfering RNA-mediated suppression of HuR in LLC-PK(1) cells increased apoptosis during energy depletion, while overexpression of HuR diminished apoptosis. Suppression of HuR also resulted in diminished levels of key cell survival proteins such as Bcl-2 and Hsp70. Furthermore, rat kidneys were subjected in vivo to transient ischemia followed by varying periods of reperfusion. Ischemia and reperfusion (I/R) affected intensity and distribution of HuR in a nephron segment-specific manner. Cells of the proximal tubule, which are most sensitive to I/R injury, demonstrated a transient shift of HuR to the cytoplasm immediately following ischemia. Over a 14-day period following the onset of reperfusion, nuclear and total HuR protein gradually increased in cortical and medullary proximal tubules, but not in non-proximal tubule cells. HuR mRNA was expressed in two forms with alternate transcriptional start sites that increased over a 14-day I/R period, and in vitro studies suggest selective translatability of these two mRNAs. Baseline and I/R-stimulated levels of HuR mRNA did not parallel those of HuR protein, suggesting translational control of HuR expression, particularly in medullary proximal tubules. These findings suggest that alterations in distribution and expression of the antiaptotic protein HuR specifically in cells of the proximal tubule effect a protective mechanism during and following I/R injury in kidney.
Assuntos
Antígenos de Superfície/metabolismo , Rim/metabolismo , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Antígenos de Superfície/genética , Apoptose/fisiologia , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Rim/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/patologia , Suínos , TransfecçãoRESUMO
Tropomyosins are coiled-coil dimers that bind to the major groove of F-actin and regulate its accessibility to actin-modifying proteins. Although approximately 40 tropomyosin isoforms have been identified in mammals, they can broadly be classified into two groups based on protein size, that is, high molecular weight and low molecular weight isoforms. Osteoclasts, which undergo rounds of polarization and depolarization as they progress through the resorptive cycle, possess an unusual and highly dynamic actin cytoskeleton. To further define some of the actin regulatory proteins involved in osteoclast activity, we previously performed a survey of tropomyosin isoforms in resting and resorbing osteoclasts. Osteoclasts were found to express two closely related tropomyosins of the high molecular weight type, which are not expressed in monocytic and macrophage precursors. These isoforms, Tm-2 and Tm-3, are not strongly associated with actin-rich adhesion structures, but are instead distributed diffusely throughout the cell. In this study, we found that Tm-2/3 expression occurs late in osteoclastogenesis and continues to increase as cells mature. Knockdown of these isoforms via RNA interference results in flattening and increased spreading of osteoclasts, accompanied by diminished motility and altered resorptive capacity. In contrast, overexpression of Tm-2, but not Tm-3, caused morphological changes that include decreased spreading of the cells and induction of actin patches or stress fiber-like actin filaments, also with effects on motility and resorption. Suppression of Tm-2/3 or overexpression of Tm-2 resulted in altered distribution of gelsolin and microfilament barbed ends. These data suggest that high molecular weight tropomyosins are expressed in fusing osteoclasts to regulate the cytoskeletal scaffolding of these large cells, due at least in part by moderating accessibility of gelsolin to these microfilaments.
Assuntos
Osteoclastos , Isoformas de Proteínas , Tropomiosina , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Movimento Celular/fisiologia , Forma Celular , Gelsolina/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Peso Molecular , Osteoclastos/citologia , Osteoclastos/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tropomiosina/química , Tropomiosina/metabolismoRESUMO
Skeletal quantity and quality are determined by processes of bone modeling and remodeling, which are undertaken by cells that build and resorb bone as they respond to mechanical, hormonal, and other external and internal signals. As the sole bone resorptive cell type, osteoclasts possess a remarkably dynamic actin cytoskeleton that drives their function in this enterprise. Actin rearrangements guide osteoclasts' capacity for precursor fusion during differentiation, for migration across bone surfaces and sensing of their composition, and for generation of unique actin superstructures required for the resorptive process. In this regard, it is not surprising that myosins, the superfamily of actin-based motor proteins, play key roles in osteoclast physiology. This review briefly summarizes current knowledge of the osteoclast actin cytoskeleton and describes myosins' roles in osteoclast differentiation, migration, and actin superstructure patterning.